Il-6 activity inhibitor

ABSTRACT

The invention relates to a nucleotide sequence, which is able to inhibit the IL-6 activity, its use in therapy as well as pharmaceutical compositions containing it. 
     In particular, it relates to a nucleotide sequence which comprises: 
     i) at least one nucleotide sequence that is an APRE element of general formula ZXMYKGKAA, wherein Z represents T or G or can also be absent, X represents T or can also be absent, M represents C or A, Y represents C or T and K represents T or G, 
     in conjunction with 
     ii) at least one nucleotide sequence constituting a transcription factor binding site other than the APRE element, such as those present in promoter regions.

FIELD OF THE INVENTION

The present invention relates to a nucleotide sequence, which is able toinhibit the IL-6 activity, its use in therapy as well as pharmaceuticalcompositions containing it.

BACKGROUND OF THE INVENTION

IL-6 is a protein belonging to the group of cytokines, which proved toplay a key role in the organism's immune response and haematopoiesisstimulation.

Many biological functions have, in fact, been found for IL-6 in thehematopoietic and lymphoid system, in the liver and in other targetorgans and cells. Some of these functions are beneficial, while othersare related to pathological states. Among the latter functions, IL-6 hasbeen found to be a growth factor for multiple myeloma cells; anti-IL-6antibodies were shown to transiently block myeloma cell proliferation ina leukemic patient (see for example Klein et al., Blood, 78, (5),pp.1198-1204, 1991 and Lu et al., Eur. J. Immunol., 22, pp. 2819-24,1992).

Elevated IL-6 levels have been correlated with autoimmune andinflammatory diseases, such as rheumatoid arthritis, glomerulonephritis,psoriasis, and Castelman's disease (see for example Graeve et al., Clin.Investig., 71, pp.664-71, 1993). IL-6 has also been shown to play adirect role in bone loss and hypercalcemia (see for example Poli et al.,Embo J., 13, (5) pp. 1189-96 and Yoneda et al., Cancer Res., 53, pp.737-40, February 1993).

The development of inhibitors of IL-6 activity has therefore been thesubject of active research. For this purpose, different approaches havebeen pursued, including the use of antibodies against IL-6 (as reportedby Klein et al. above), gp130 or gp80; the use of soluble gp130; or theuse of muteins for IL-6, or IL-6 Receptor.

Since these approaches might be associated with specific unwantedeffects in clinical applications (as reported by Lu et al., above), thesetting-up of additional strategies to inhibit IL-6 activity would beuseful.

The Applicant has, therefore, investigated a different approach toinhibit IL-6 activity: by blocking the intracellular proteins mediatingthe IL-6 signal.

The transduction of the IL-6 signal in responsive cells has beenintensively investigated. Fowlkes et al. (PNAS USA, 81, pp. 2313-6,1984) first suspected DNA responsive elements specific for IL-6 flankingthe rat fibrinogen genes.

Later on, Kunz et al. (Nuc. Ac. Res., 17, (3), 1121-37, 1989) showed aresponsive element with a core sequence identical to that of ratfibrinogen genes (CTGGGA) to respond to IL-6 in the rat α₂-macroglobulin gene.

DNA responsive elements with sequences related to those above-mentionedhave also been defined in the genes coding for the human C ReactiveProtein (CRP) (see Toniatti et al., Mol. Biol. Med, 7, pp. 199-212,1990), human haptoglobin (see Oliviero et al., Embo J. 6, (7), pp.1905-12, 1987) and in other genes coding for additional acute phaseproteins induced by IL-6 (see Heinrich et al., Biochem. J., 265, pp.621-36, 1990), leading to the definition of a core consensus sequenceCTGGGAW or CTGGRAA, where W stands for A or T, and R stands for A or G.

Hocke et al. (Mol. Cell. Biol., 12, (5), pp. 2282-94, 1992) indicatedthat multiple related core sequences, similar to the core sequencementioned above, might be present in regulatory regions of wild-typegenes responding to IL-6 and that this multiplicity leads toamplification of the response, as functionally analyzed with a reportergene assay.

Wegenka et al. (Mol. Cell. Biol., 13, (1), pp. 276-88, 1993) haverecently indicated an enlarged version of the core consensus sequence asthe Acute Phase Response Element (APRE), active in hepatoma cells thatcan be represented by the formula KTMYKGKAA, wherein M stands for C orA, K stands for T or G, Y stands for C or T.

By Yuan et al. (Mol. Cell. Biol., 14, (3), pp. 1657-68, 1994) it hasbeen shown that such APRE-like sequences bind a protein transcriptionfactor having a molecular weight of about 90 KD, called APRF which hasbeen recently cloned (see Akira et al., Cell, 77, pp. 63-71, 1994). Inpractice, the binding of activated APRF to APRE sequences wouldtherefore lead to activation of IL-6-inducible genes (containing suchAPRE sequences) in IL-6-responsive cells.

As a consequence of this, an APRE element can be used as enhancer of atarget gene in IL-6 responsive cells in the following way: in IL-6responsive cells, the treatment with IL-6 induces the synthesis of APRFproteins, which bind to the APRE element, and such binding activates theexpression of the target gene.

Serlupi Crescenzi et al. (Poster at the 12th European Imunol., Meeting,Barcelona, Jun. 14-17, 1994) have shown that an 8-fold repetition of theAPRE DNA sequence (M8) is responsible for a 50-100 fold induction byIL-6 of a reporter gene in HepG2 human hepatoma cells.

SUMMARY OF INVENTION

The main object of the present invention is a nucleotide sequence whichis able to inhibit the IL-6 activity, that comprises:

i) at least one nucleotide sequence that is an APRE element of generalformula ZXMYKGKAA, wherein Z represents T or G or can also be absent, Xrepresents T or can also be absent, M represents C or A, Y represents Cor T and K represents T or G,

in conjunction with

ii) at least one nucleotide sequence constituting a transcription factorbinding site other than the APRE element, such as those present inpromoter regions.

Examples of these latter type of nucleotide sequences include: TATA box,and the binding sites for transcription factors, such as AP-1 (seeRiabowol et al., PNAS USA, 89, pp, 157-61, 1992), AP-2, HNF-1 (seeClusel et al., Nuc. Ac. Res., 21 (15), pp. 3405-11), SP-1 (see Wu etal., Gene, 89, pp. 203-9, 1990), NF-icB (see Bielinska et al., Science,250, pp. 997-1000, 1990), Oct-1, E-2 and SRF transcription factors.

In a preferred embodiment of the present invention, both the APREelement (i) and/or the nucleotide sequence (ii) of the above generalformula are each repeated at least 2 times, more preferably, from 3 to10 times, still more preferably 8 times.

In a further preferred embodiment of the present invention, the element(i) comprises at least two different APRE elements and/or the nucleotidesequence (ii) comprises at least two different oligonucleotide sequencesconstituting a transcription factor binding site other the APRE element.

The nucleotide sequence (ii) is preferably the SV40 early promoter.

The APRE element (i) comprises, for example, the following nucleotidesequence: TTCTGGGAA.

FIG. 2 reports a nucleotide sequence which falls within the scope of theinvention, in accordance with its preferred embodiments. Such nucleotideis also reported as SEQ ID NO: 1 and it constitutes one object of theinvention.

A further object of the present invention is a plasmid vector containingthe nucleotide sequence of the invention.

An additional aspect of the present invention is the use of thenucleotide sequence of the invention as a therapeutic tool to inhibitthe action of IL-6, in those conditions where IL-6 plays a pathologicalrole.

The present invention also provides pharmaceutical compositionscomprising a nucleotide sequence or a plasmid vector according to theinvention. Such compositions can be formulated for oral, rectal,intravenous or topical use. Formulations of the present invention mayinclude the use of any combination of viral-mediated gene transfer,liposome formulation, receptor-mediated DNA delivery and/or dumbbellstructures of the active nucleotide inhibitory sequence according to theinvention.

The inhibitory action of the nucleotide sequence of the invention hasbeen determined with a reporter gene assay.

The reporter gene assay of the invention is based on the ability of theAPRE element to function as enhancer of any gene in IL-6 responsivecells (as reported above). In this case, the APRE element is used asenhancer of a target reporter gene (which is, for example, theLuciferase gene) in IL-6 responsive cells (which are, for example,hepatic cells such as HepG2). The cells are then treated with IL-6: ifenough of the APRF proteins produced are captured by an excess of thenucleotide sequence of the invention, the target gene will not beactivated.

According to this assay, inhibitor plasmids are constructed, whichcontain the nucleotide sequence of the invention. An example of suchplasmids is reported in FIG. 1 together with its construction strategy.

This and other plasmids, containing the nucleotide sequence of theinvention, are also intended to constitute a further embodiment of theinvention.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Construction strategy of plasmid pM8SV. pM8SVL is digested withSal I and Hind III and the sticky ends are transformed in blunt ends bythe Kleenow reaction. The resulting 3.2 Kb DNA fragment is purified byagarose gel electrophoresis, then it is self-ligated Plasmid pM8SV isthus generated, containing the M8 sequence (about 170 bp) and thesequence from the SV40 virus early promoter (about 190 bp), but lackingthe luciferase gene.

FIG. 2. Sequence of the BamH I-Hind III inhibitor DNA fragment ofplasmid pM8SV. The continuous line above the upper part of the shownnucleotide sequence represents the M8 sequence. The bold continuous lineabove the lower part of the nucleotide sequence represents the SV40promoter sequence. BamH I and Hind III restriction sites are alsoindicated.

FIG. 3. IL-6 reporter gene assay in T47D and M1 cells. Test of differentM8-containing plasmids. Reported values of light emission are averagesof duplicate determinations of cps integrated over a period of 30seconds (AUC=Area Under the Curve) from transfected cells. Each barrepresents the average of three transfections ±SEM.

FIG. 4. Time-course of luciferase inducibility of transfected Hep G2cells after treatment with IL-6. Plasmid pM8SVL was used as the positivereporter gene plasmid. Each bar represents the average of twotransfections ±SEM. Two experiments are shown in the Figure.

FIG. 5. HepG2 reporter gene assay for IL-6. Test of pM8 as inhibitorplasmid against pM8SVL as reporter gene plasmid. Each bar represents theaverage of three transfections ±SEM. Four experiments are shown in theFigure.

FIG. 6. HepG2 reporter gene assay for IL-6. Test of pN8SV as inhibitorplasmid against pN8SVL as reporter gene plasmid. each bar represents theaverage of three transfections ±SEM. Results are expressed as % ofinhibition with respect to HepG2 cells transfected with carrier plasmidpC without inhibitor plasmid.

FIG. 7. HepG2 reporter gene assay for IL-6. Test for pSV and pM8SV asinhibitor plasmids against pN8SVL reporter gene plasmid. Each barrepresents the average of three transfections ±SEM. Results areexpressed in % of inhibition with respect to HepG2 cells transfectedwith carrier plasmid pC without inhibitor plasmid.

FIG. 8. inhibition test of IL-6 activity by inhibitor plasmids pM8 andpM8SV in a HepG2 reporter gene assay based on the luciferase gene undercontrol of the IL-6 inducible regulatory sequences from the humanhaptoglobin gene promoter. Each bar represents the average of threetransfections ±SEM Results are expressed in % of inhibition with respectto HepG2 cells transfected with carrier plasmid pC without inhibitorplasmid. Transfections were performed with 0.1 μg of reporter plasmidDNA and the shown molar excess of inhibitor plasmid. The total amount oftransfected DNA was kept constant with the carrier plasmid. Transfectedcells were treated for 18 hours with 1 ng/ml of IL-6.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Construction of plasmids. An IL-6-responding luciferase reporter geneplasmid was constructed by first preparing through chemical synthesis adouble-stranded oligonucleotide of 38 bp with an uncut BamH I site atits 5' blunt end, and a protruding lower strand of 4 nucleotidescompatible with the Bgl II and BamH I sites at the 3' end. Thissynthetic oligonucleotide was named M2 and it contained two identicalAPRE sequences from the rat α2-macroglobulin gene promoter region. Thesequence of the oligonucleotide (upper strand, 5' to 3') was as follows:

GGATCCTTCTGGGAATTCTGATCCTTCTGGGAATTCTG (SEQ ID NO: 2). Thisoligonucleotide was cloned in the Sma I-Bgl II sites of plasmid pGL2-pv(from Promega Corporation), where the expression of the luciferasereporter gene is driven by the SV40 virus early promoter, thus forming,after self-ligation, plasmid pM2SVL.

The synthetic oligonucleotide, through its 5' blunt end, was alsoligated to the Sma I site of the same plasmid pGL2-pv and the resultinglinear ligation product was then cut with Hind III. The resulting linearvector was used as a recipient to clone the following DNA fragments: 1)the BamH I-Hind III fragment of 238 bp from plasmid pM2SVL, thusforming, after self-ligation, plasmid pM4SVL; 2) the BamH I-Hind IIIfragment of 280 bp from pM4SVL, thus forming, after self-ligation,plasmid pM6SVL and 3) the BamH I-Hind III fragment of 322 bp frompM6SVL, thus forming, after self-ligation, plasmid pM8SVL.

Plasmid pM8L was constructed by digesting pM8SVL with Sfan I and byconverting the sticky ends in blunt ends by fill-in reaction with theKleenow enzyme. After BamH I digestion, the resulting M8 DNA fragment of163 bp was ligated with a 16 bp BamH I-Kpn I synthetic adapter andcloned in a 5.6 Kb DNA fragment resulting from Sma I+Kpn I digestion ofthe pGL2-b vector (from Promega Corporation). Plasmid pGL-2b isidentical to the above-mentioned pGL2-pv plasmid, except for the absenceof the SV40 promoter sequence in pGL2-b. The 16 bp adapter contained amultiple cloning site, and it was prepared by chemical synthesis withthe following sequence:

upper strand: ⁵ 'CGCGGCCGCCTCGAGG³ ' (SEQ ID NO: 3);

lower strand: ⁵ 'GATCCCTCGAGGCGGCCGCGGTAC³ ' (SEQ ID NO: 4).

The plasmid resulting from the above construction was pM8L, and it hadthe M8 DNA sequence without promoter, embedded in a multiple cloningsite, upstream to the luciferase gene.

The luciferase reporter gene plasmid pM8TKL was prepared by cuttingplasmid pGEM-TK-CAT (described in Cohen et al., EMBO J., 7(5), pp.1411-9, 1988) with Xba I and Bgl II. The resulting 181 bp fragmentcontaining the TK promoter sequence of the viral HSV thymidine kinasegene was ligated with the 5.8 Kb Nhe I-Bgl II fragment of the pM8Lvector, between the M8 and the luciferase DNA sequences.

The luciferase reporter gene plasmid pHPSVL was constructed first by PCRamplification of 841 bp from the haptoglobin gene promoter region fromhuman genomic DNA. The 3' end of the amplified PCR fragment correspondedto position -36 from the transcription start of the human haptoglobingene (as reported in Maeda et al., J. Biol. Chem., 260(11), pp.6698-709, Jun. 10, 1985). This PCR product was prepared with upper andlower primers containing respectively Mlu I and Bgl II restrictionsites. The DNA primers synthesised for genomic amplification of thehaptoglobin promoter region had the following sequence:

upper primer: ⁵ 'CTACGCGTGCAGTATTGACCCTTCCTCCT³ ' (SEQ ID NO: 5);

lower primer: ⁵ 'CGCAGATCTAGCTCACTTCTCCCCCTTC³ ' (SEQ ID NO: 6).

The PCR fragment thus obtained was inserted in Mlu I and Bgl II sites ofthe luciferase reporter gene plasmid pGL2-pv mentioned above, upstreamto the SV40 early promoter. The resulting plasmid was pHPSVL.

In order to construct the inhibitor plasmid pM8, the above plasmid pM8Lwas digested with Sal I and Bgl II and the sticky ends thus generatedwere repaired in blunt ends by the Kleenow reaction. The resultingfragment of 3.1 Kb, lacking the luciferase gene sequence, was purifiedby agarose gel electrophoresis, then it was self-ligated to generate theinhibitor plasmid pM8.

The inhibitor plasmid pM8SV was prepared as shown in FIG. 1. pM8SVL wasdigested with Sal I and Hind III and the sticky ends were transformed inblunt ends by the Kleenow reaction. The resulting 3.2 Kb DNA fragmentwas purified by agarose gel electrophoresis, then it was self-ligated.Plasmid pM8SV was thus generated, containing the M8 sequence (about 170bp) and the sequence from the SV40 virus early promoter (about 190 bp),but lacking the luciferase gene.

The inhibitor plasmid pSV was constructed by cutting out the Sal I-HindIII fragment containing the luciferase gene from plasmid pGL2-pvmentioned above. The resulting fragment of 3.1 Kb was subject to fill-inreaction with the Kleenow enzyme, purified by agarose gelelectrophoresis and then it was self-ligated, thus yielding plasmid pSVwhich contains the SV40 promoter but lacks the luciferase gene.

The carrier plasmid pC was prepared by cutting plasmid pGL2-b mentionedabove with Sal I and Hind III restriction enzymes. The resultingfragment of 2.9 Kb was subject to fill-in reaction with the Kleenowenzyme, purified by agarose gel electrophoresis and then it wasself-ligated, thus yielding plasmid pC.

All plasmid constructs described above were used to transform the E.Coli strain XL1-Blue with standard techniques (Ausubel R. et al.,Current Protocols in Molecular Biology. Greene Publishing Associates andWiley Interscience, New York). Plasmid DNA was extracted fromtransformed clones by minipreparative alkaline lysis method (accordingto Ausubel, above). Plasmid DNA was controlled by restriction analysisand by agarose gel electrophoresis. Clones with the expected patternwere selected. To obtain purified plasmid preparations to be used intransfection of mammalian cells, 300 ml cultures of the selected E. ColiXL1-Blue transformants were prepared. The plasmids were then purified byQIAGEN tip 500 ion-exchange minicolumns, by following the manufacturerinstructions.

EXAMPLE 2

Cell lines and culture conditions. HepG2 human hepatoma cells (ATCC)were cultured in MEM supplemented with 10% FCS, 5 mM L-glutamine, 20 mMHEPES, 100 U/ml penicillin/streptomycin. T47D human breast carcinomacells (ATCC) were cultured in DMEM supplemented with 10% FCS, 5 mML-glutamine, 20 mM HEPES, 100 U/ml penicillin/streptomycin. M1 mousemyeloid leukaemia cells (ATCC) were cultured in RPMI supplemented with10% FCS, 5 mM L-glutamine, 20 mM HEPES, 100 U/mlpenicillin/streptomycin. Culture of the above cell lines was performedin the presence or absence of the appropriate dose of IL-6 (CHO-derivedh IL-6 from Interpharm Laboratories), as specified below.

EXAMPLE 3

Transient transfections and luciferase and β-galactosidase assays.Transient transfection of HepG2 human hepatoma cells and T47D humanbreast carcinoma cells was carried out using calcium phosphate-DNAprecipitation in hepes buffer, according to standard procedures (AusubelR. et al., Current Protocols in Molecular Biology. Greene PublishingAssociates and Wiley Interscience, New York). Transient transfection ofmouse M1 myeloid leukaemia cells was performed using DEAE-dextran,according to standard procedures (according to Ausubel, above).

In order to detect luciferase and β-galactosidase activities, cells wereextracted in situ by incubation for 15 minutes at room temperature with1 ml/10⁶ cells of extraction buffer (25 mM TRIS-phosphate pH 7.8, 2 mMDTT, 2 mM EDTA, 10% glycerol, 1% Triton X-100). For luciferase activity,20 μl of cell extract were directly assayed with 100 μl of luciferaseassay buffer (20 mM Tricine, 1.07 mM (MgCO₃)₄ Mg(OH)₂.5H₂ O, 2.67 mMMgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 0.27 mM CoenzymeA, 0.47 mM luciferin,0.53 mM ATP). Forβ-galactosidase activity, 10 μl of cell extract wereincubated for 1 hour at 37° C. with 100 μl of Lumigal substrate (fromLumigen). Lumigal and luciferase readings were performed with a BertholdAutolumat LB953 luminometer, the output being counts per seconds (cps)integrated over a period of 30 seconds for luciferase and 15 seconds forβ-galactosidase.

Reporter plasmid pM8SVL was shown to confer IL-6 responsiveness aftertransient transfection of HepG2 cells, by increasing up to 50-100 timesthe expression of luciferase activity (Serlupi Crescenzi et al., Posterat the 12th European Immunol., Meeting, Barcelona, Jun. 14-17, 1994).

This plasmid was also tested in T47D human breast carcinoma cells and inmouse M1 myeloid leukaemia cells. M1 cells were transfected with 0.5 μgDNA/10⁶ cells using DEAE/Dextran, while 2.5 μg DNA/10⁵ cells were usedfor calcium phosphate transfection of T47D cells.

Only very limited features are shared by these two cell lines, apartfrom their common response to human IL-6. Results (FIG. 3) showed thathe M8 DNA molecule in pM8SVL plasmid was significantly active in bothell lines after IL-6 treatment. As shown in FIG. 3, a significantresponse to IL-6 was also observed in M1 cells with the reporter geneplasmid pM8TKL, where the M8 molecule was flanked by the thymidinekinase promoter, which is different from the SV40 promoter present inpM8SVL.

Time-course of luciferase inducibility of transfected HepG2 cells hasbeen tested after treatment with IL-6 (1 ng/ml). Plasmid pM8SVL was usedas positive reporter gene plasmid (at 0.2 μg/10⁵ cells). Cells weretransfected overnight, splitted and then exposed to the IL-6 treatment.The results are reported in FIG. 4 and they show that almost fullresponse to IL-6 could be achieved after only two hours of IL-6treatment.

EXAMPLE 4

Test of inhibitor plasmid pM8. Inhibition of IL-6 activity by M8 DNAmolecules was measured after co-transfection in HepG2 cells of i) thepM8SVL reporter gene plasmid responding to IL-6 and ii) the M8 moleculeinserted in the pM8 inhibitor plasmid. This latter plasmid hosts the M8sequence but it has not the ability to confer responsiveness to IL-6.HepG2 cells were transfected with up to 2.5 μg/10⁵ cells of theIL-6-responding reporter plasmid pM8SVL (containing M8 and theluciferase gene) and 10 or 50 fold molar excess of pM8, in the presenceof various doses of IL-6. The total amount of DNA per transfection waskept constant with the use of the carrier plasmid pC, which is identicalto pM8, except for the absence in the former plasmid of the specific 165bp-long M8 DNA fragment.

As reported in FIG. 5, the inhibitor plasmid did not show significantand reproducible specific inhibition of the IL-6 activity in fourexperiments, even at 50 fold molar excess of the pM8 inhibitor plasmid.In these experiments the IL-6-responding reporter gene plasmid pM8SVLwas used at a dose of 0.1 μg/10⁵ cells, while the constant amount oftotal DNA used in transfection was 5 μg/10⁵ cells. The suboptimal doseof IL-6 used in these experiments was 1 ng/ml. As shown in FIG. 5,variability was acceptable with these experimental conditions, given thefact that distinct transfections per se are an inevitable source ofvariability.

EXAMPLE 5

Test of inhibitor plasmid pM8SV. The results reported in FIG. 5 weresomewhat surprising, because the active M8 inhibitor DNA sequence of theinhibitor plasmid pM8 is identical to the active sequence of the pM8SVLreporter gene plasmid. After co-transfection, competition between thesetwo identical M8 sequences present in different plasmids would thereforebe expected, resulting in inhibition of IL-6 activity in the reportergene assay.

Moreover, the results described above can not be explained by thepresence of an excess of activated, IL-6-specific transcriptionfactor(s), which are not sufficiently neutralised by the M8 inhibitorDNA molecules, since the data were obtained in the presence of alimiting amount of IL-6. In addition, published data on similarexperiments with DNA binding sites for known transcription factors(26-30) are not consistent with the results reported in FIG. 5.

An alternative explanation for the results shown in FIG. 5 could be thatsequences other than M8 in the reporter gene plasmid may contribute tothe IL-6-specific signal transduction. These sequences should be missingin the pM8 inhibitor plasmid, but they should be present in the positivereporter gene plasmid pM8SVL (e.g., sequences of the SV40 early genepromoter, which can bind general transcription factors). The inhibitorplasmid pM8 would therefore be ineffective in competing with thereporter gene plasmid pM8SVL.

In order to test this hypothesis, an inhibitor plasmid (pM8SV) wasconstructed, containing as IL-6-inhibitor DNA sequences, both the M8sequence and the SV40 promoter sequence (see FIG. 1). This inhibitorplasmid was tested in the IL-6 reporter gene assay with HepG2 cells.Four independent experiments were performed, with duplicatetransfections per experiment. Transfections were performed with 0.1 μgof reporter plasmid DNA and the molar excess of inhibitor plasmid shownin FIG. 6. The total amount of transfected DNA was kept constant withthe carrier plasmid Transfected cells were treated for 18 hours with 1ng/ml of IL-6.

Results, shown in FIG. 6, indicated a clear dose-dependent inhibition ofIL-6 activity by the pM8SV inhibitor plasmid. Raw data of FIG. 6 arereported are reported on Table 1. Three replicate transfections per doseof inhibitor plasmid were performed in each experiment. For eachexperiment, the induced and non-induced values shown in a single rowcome form the same transfection. Reported values of light emission arecps integrated over a period of 30 seconds. As it can be seen from suchTable 1, some variability was observed in these experiments, especiallyat lower doses of the inhibitor plasmid, but usually CVs of replicatetransfections were well below 20%.

In order to rule out that the inhibition of IL-6 activity provided bythe pM8SV inhibitor plasmid was due exclusively to the SV40 promoter DNAsequence and not to the combination of M8 and SV40 sequences, anadditional inhibitor plasmid was constructed and tested in the reportergene assay. This plasmid contained only the SV40 DNA sequence asinhibitor of IL-6 activity, without the M8 inhibitor sequence, nor theluciferase gene.

Transfections were performed with 0.1 μg of reporter plasmid DNA and themolar excess of inhibitor plasmid shown in FIG. 7. The total amount oftransfected DNA was kept constant with the carrier plasmid. Transfectedcells were treated for 18 hours with 1 ng/ml of IL-6. The results,reported in FIG. 7, show that this inhibitor plasmid (pSV) displayedonly partial inhibition of IL-6 activity, which never resulted to beabove 40% and was not dose-dependent. This allows to conclude that theSV40 DNA sequence alone was not sufficient for effective inhibition ofIL-6 activity. On the other hand, the luciferase-containing reporterplasmid pCL2-pv contains the SV40 promoter sequence but not the M8sequence, thus resulting in a basal level of luciferase expression notfurther inducible by IL-6. In this plasmid, the basal level ofluciferase expression was not inhibited by the pM8SV inhibitor plasmid,thus showing that transcription factors which are specifically boundonly to the SV40 promoter region of pGL2-pv, are not effectively removedby the pM8SV inhibitor plasmid. In fact, in the presence of the latterinhibitor plasmid, luciferase activity from the reporter plasmid pGL2-pvwas even higher than in the absence of the inhibitor plasmid (notshown).

EXAMPLE 6

Inhibition by pM8SV of the IL-6 activity conferred by the reporter geneplasmid pHPSVL. We then wanted to test the inhibitor plasmid pM8SV in anadditional reporter gene assay for IL-6, where the target DNA sequencemediating the IL-6 signal in the reporter gene plasmid is not perfectlymatching the M8 inhibitor sequence. HepG2 cells were thereforetransfected with plasmid pHPSVL, which contains 841 base pairs from thepromoter sequence of the human haptoglobin gene, flanked by the SV40promoter and the luciferase gene. One APRE site from the haptoglobinpromoter sequence (according to Maeda et al., J. Biol. Chem., 260(11),pp. 6698-709, Jun. 10, 1985) is present in this plasmid. We havepreviously shown that this plasmid does respond to IL-6 by a 6-8-foldincrease of luciferase expression (Serlupi Crescenzi et al., Poster atthe 12th European Immunol., Meeting, Barcelona, Jun. 14-17, 1994).

Results from one experiment of triplicate transfections are shown inFIG. 8. After co-transfection of the reporter gene plasmid pHPSVL with50-fold molar excess of the carrier plasmid, inducibility by IL-6resulted in about 4-fold higher luciferase expression with respect tothe basal level. On the contrary, co-transfection with 50-fold molarexcess of the inhibitor plasmid pM8SV completely abolished theinducibility by IL-6. The pM8SV inhibitor plasmid was therefore able toinhibit IL-6 activity also when an IL-6-responding reporter gene plasmiddifferent from pM8SVL was tested (e.g., the reporter plasmid pHPSVL).

EXAMPLE 7

Test of inhibitor plasmid pM8SV in T47D cells. The pM8SV inhibitorplasmid was also tested in the pM8SVL reporter gene assay, using T47Dhuman breast carcinoma cells, where the transduction of the IL-6 signalmight be different than in hepatoma cells. As previously mentioned, theIL-6 reporter gene assay with the pM8SVL reporter gene plasmid isworking on this cell line, although it is not optimised. Sincesensitivity of the assay is somewhat lower with T47D cells,transfections were performed with 1 μg of the positive reporter geneplasmid/10⁵ cells, which is a relatively high level of plasmid DNA.Non-specific inhibition was therefore observed in this experiment in thepresence of excess carrier or inhibitor plasmid, resulting in highervariability of the IL-6-specific inhibition.

The results are shown in Table 2. Averages (AVG) and standard deviations(SD) of triplicate transfections from two experiments are shown.

Average values of fold induction are calculated from the raw values ofeach transfection. 1 and 0.5 μg of reporter gene plasmid were used inexperiments 1 and 2 respectively per 10⁵ transfected cells.

Separate transfections of carrier plasmid and inhibitor plasmids wereperformed in these experiments, both plasmids being used at theindicated molar excess with respect to the reporter plasmid.

After transfection cells were induced for 18 hours with 1 ng/ml of IL-6.Reported values of light emission are cps integrated over a period of 30seconds. IL-6 specific inhibition in these experiments was evaluated bycomparing the induction obtained in the presence of excess of inhibitorplasmid with the induction obtained in the presence of the correspondingdose of carrier plasmid.

Moreover, because of non-specific inhibition, the basal level ofluciferase expression was close to the quantitation limit of the assay.Results, reported in Tab. 2 (see Experiment 1) showed that, aftertriplicate transfections, relevant, specific inhibition of IL-6 activitywas obtained at 20-fold molar excess of inhibitor plasmid pM8SV, but notat 10-fold molar excess. A 50-fold molar excess of the inhibitor plasmidcould not be tested in this experiment, because non-specific inhibitionbecame too high.

These results could not be reproduced in an additional experiment, when0.5 μg DNA/10⁵ cells were used (Tab. 2, see Experiment 2). The reasonfor this lack of reproducibility can be due to the relatively highvariability and low sensitivity of the T47D reporter gene assay.Alternatively, differential target cell selectivity could explain theseresults.

EXAMPLE 8

Definition of the minimal DNA inhibitory sequence. In order to identifythe minimal DNA sequence which retains the ability to inhibit thebinding of transcription factors relevant for IL-6 inducibility, theexperimental approach of electrophoretic mobility shift assay (EMSA) canbe used (according to Ausubel). A test for functional inhibitionimparted by this minimal sequence can be set using the reporter geneassay mentioned in Example 3 and following Examples. The miimal DNAsequence we have shown to functionally inhibit IL-6 activity in areporter gene assay was the BamH I-Hind III fragment of 350 bp of theinhibitor plasmid pM8SV, which contains an 8-fold repetition of an APREDNA sequence and the SV40 early promoter sequence. This latter sequenceis known to contain binding sites for general transcription factors suchas an AP-1-like site (as reported in Zenke et al., EMBO J., 5(2), pp.387-97, 1986) and a 6-fold repetition of the Sp-1 site (as reported inDynan et al., Cell, 35, pp. 79-87, 1983). The BamH I-Hind III DNAfragment can be deleted at its 5' and/or 3' ends, by conventional meanssuch as PCR or nuclease treatment (according to Ausubel) in order tocontain, e.g., two APRE sequences and only a single or a few bindingsite for specific transcription factors from the SV40 early promoter.The resulting DNA sequences can be used for inhibition tests of thepSVM8L-based reporter gene assay in HepG2 cells.

Furthermore, a minimal DNA fragment which retains full ability tofunctionally inhibit the IL-6 signal can be used in EMSA, by labellingthis DNA with a ³² P nucleotide through conventional means, such asend-labelling or fill-in Kleenow reactions. The resulting labelled DNAfragment can be incubated with nuclear extracts from HepG2 cells afterIL-6 treatment.

Increasing amounts of a competitor DNA sequence can also beco-incubated, such as anyone of the unlabelled DNA fragments whichresulted from the above-mentioned deletions of the BamH I-Hind IIIinhibitor DNA fragment. After incubation, the mixture can be run innon-denaturing polyacrylamide gel electrophoresis. The binding ofrelevant transcription factors to the tested labelled DNA fragment willbe revealed by a shift in the gel mobility (retardation) expected forthe unbound, labelled DNA The inhibition of this binding in the presenceof competitor, unlabelled DNA sequences will be revealed by the specificdisappearance of the shifted gel bands.

                                      TABLE 1                                     __________________________________________________________________________    HepG2 reporter gene assay for IL-6.                                            Test of pSVM8 inhibitor plasmid against pSVM8L reporter gene plasmid         Fold mol.                                                                                                             excess of Experiment 1 Experiment                                            2 Experiment 3 Experiment 4            inhibitor                                                                           - IL-6                                                                             + IL-6                                                                              - IL-6                                                                             + IL-6                                                                              - IL-6                                                                             + IL-6                                                                              - IL-6                                                                              + IL-6                           __________________________________________________________________________    0     30.330                                                                             1.562.000                                                                            49.200                                                                            1.664.000                                                                           30.880                                                                             2.489.000                                                                           37.430                                                                              1.587.000                           34.760 1.227.000  83.590 1.914.000 47.610 2.266.000 34.000 1.433.000                                                      32.920 1.711.000  91.560                                                    1.754.000 49.670 3.239.000                                                    31.130 1.362.000                   10 29.400   751.800 144.300 2.380.000 33.680   923.100 32.390   476.200        34.660   951.300 161.500 2.202.000 35.020   722.500 39.610   790.400                                                      24.530 1.159.000 129.200                                                    1.953.000 37.620 1.110.000                                                    32.770   516.900                   25 40.730   568.100  85.410   745.300 46.760   538.400 36.790   298.100        45.320   677.700  61.340   671.600 33.520   414.000 20.320   255.500                                                      32.670   620.400  71.040                                                    773.300 27.950   543.500                                                      26.080   318.800                   50 53.210   312.200 148.700   857.900 23.820   173.500 120.800                                                           586.000                             60.110   268.500 159.700   655.300 25.370   150.500 96.900   716.400                                                      73.810   427.300  68.630                                                    517.500 46.470   287.500                                                      195.800  1.029.000               __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    T47D reporter gene assay for IL-6                                             Fold molar excess of inhibitor or                                                            10       20                                                    carrier plasmid                                                                              AVG (SD) AVG (SD)                                              __________________________________________________________________________    EXP. 1                                                                            Reporter                                                                             -IL-6                                                                             7832                                                                              (3795)                                                                             932 (141)                                                plasmid                                                                       plus +IL-6 423746 (108679) 58950 (29703)                                      carrier Fold 61 (27) 68 (44)                                                  plasmid Induct.                                                               Reporter -IL-6 7498 (2848)                                                    plasmid                                                                        plus   +IL-6                                                                             570099                                                                            (29927)                                                                            not determined                                            pM8 inhib.                                                                           Fold                                                                              85  (34)                                                          plasmid Induct.                                                               Reporter -IL-6 925 (123) 704 (103)                                            plasmid                                                                       plus +IL-6 98766 (21586) 11416 (11533)                                        pM8SV inhib. Fold 110 (39) 15 (13)                                           EXP. 2 pl. Induct.                                                             Reporter -IL-6 35320 (20227) 2325 (1019)                                      plasmid                                                                       plus +IL-6 390400 (45230) 77617 (30495)                                       carrier Fold 15 (11) 35 (8)                                                   plasmid Induct.                                                               Reporter -IL-6 7498 (2256)                                                    plasmid                                                                        plus   +IL-6                                                                             570099                                                                            (40806)                                                                            not determined                                            pM8 inhib.                                                                           Fold                                                                              85  (14)                                                          plasmid Induct.                                                               Reporter -IL-6 925 (495) 704 (103)                                            plasmid                                                                       plus +IL-6 98766 (64852) 11416 (11533)                                        pM8SV inhib. Fold 110 (8) 15 (13)                                             pI. Induct.                                                                __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 356 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - CCCAGGATCC TTCTGGGAAT TCTGATCCTT CTGGGAATTC TGATCCTTCT GG -            #GAATTCTG     60                                                                 - - ATCCTTCTGG GAATTCTGAT CCTTCTGGGA ATTCTGATCC TTCTGGGAAT TC -            #TGATCCTT    120                                                                 - - CTGGGAATTC TGATCCTTCT GGGAATTCTG ATCTGCATCT CAATTAGTCA GC -            #AACCATAG    180                                                                 - - TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CA -            #TTCTCCGC    240                                                                 - - CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG GC -            #CTCTGAGC    300                                                                 - - TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AA - #GCTT            356                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - GGATCCTTCT GGGAATTCTG ATCCTTCTGG GAATTCTG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CGCGGCCGCC TCGAGG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - GATCCCTCGA GGCGGCCGCG GTAC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - CTACGCGTGC AGTATTGACC CTTCCTCCT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - CGCAGATCTA GCTCACTTCT CCCCCTTC         - #                  - #                 28                                                                    __________________________________________________________________________

We claim:
 1. A nucleotide sequence which inhibits IL-6 activity, thatcomprises:i) at least one nucleotide sequence that is an APRE element ofgeneral formula ZXMYKGKAA, wherein Z represents T or G or can also beabsent, X represents T or can also be absent, M represents C or A, Yrepresents C or T and K represents T or G; ii) at least one nucleotidesequence constituting a transcription factor binding site other than theAPRE element.
 2. The nucleotide sequence according to claim 1, wherein(i) is the nucleotide sequence TTCTGGGAA.
 3. The nucleotide sequenceaccording to claim 1, wherein (ii) is selected from the group consistingof TATA Box, binding site for transcription factor AP-1, binding sitefor transcription factor AP-2, binding site for transcription factorHNF-1, binding site for transcription factor SP-1, binding site fortranscription factor NF-KB, binding site for transcription factor Oct-1,binding site for transcription factor E-2, and binding site fortranscription factor SRF.
 4. The nucleotide sequence according to claim1, wherein the APRE element (i) is repeated at least 2 times.
 5. Thenucleotide sequence according to claim 4, wherein the APRE element isrepeated from 3 to 10 times.
 6. The nucleotide sequence according toclaim 4, wherein the APRE element is repeated 8 times.
 7. The nucleotidesequence according to claim 1, wherein the sequence (i) comprises atleast two different APRE elements.
 8. The nucleotide sequence accordingto claim 1, wherein the sequence (ii) comprises at least two differentoligonucleotide sequences constituting a transcription factor bindingsite.
 9. The nucleotide sequence according to claim 8, wherein thesequence (ii) is the SV40 early promoter.
 10. The nucleotide sequenceaccording to claim 1, as reported in SEQ ID NO:
 1. 11. A plasmid vectorcontaining the nucleotide sequence of claim
 1. 12. A compositioncomprising the nucleotide sequence according to claim 1 together withone or more pharmaceutically acceptable carriers and/or excipient.
 13. Acomposition comprising the plasmid according to claim 11 together withone or more pharmaceutically acceptable carriers and/or excipients. 14.A method for inhibiting IL-6 activity, comprising the step ofintroducing the nucleotide sequence of claim 1 into cells to inhibitIL-6 activity in the cells.